Heavy metal ions play essential roles in many biological processes but can exert toxic effects at elevated concentrations. Although a variety of genetic disorders in humans such as Menkes' and Wilson's disease are known to involve disorders in heavy metal metabolism, the fundamental chemical and biological processes responsible for maintaining intracellular metal ion concentrations are not well enough understood to allow efficient intervention. The broad goal of the basic research projects described here is to elucidate the regulatory mechanisms involved in cellular responses to heavy metal stress at the macromolecular and coordination chemistry levels in well defined systems. Metal-responsive expression of both the copper detoxification genes in enteric bacteria and the metallothionein genes in mammalian cell cultures will be examined. The specific metal-receptors and other cellular factors involved in transduction of the inorganic signals will be isolated in quantities sufficient for biophysical and chemical characterization. The isolation of the eukaryotic transcription factor will be facilitated by application of a novel protein-DNA crosslinking strategy which is under development and which may be applicable to other metalloregulatory systems. The synthesis and characterization of a series of bimetallic reagents capable of reversible protein-DNA crosslinking is outlined. These reagents, based in part on the DNA-binding activity of the anticancer drug cis-diamminedichloroplatinum(II), join a growing class of inorganic tools designed as probes of biopolymer structure and function. In the case of the bacterial copper resistance system, one of the metalloregulatory genes, pcoR, has been cloned, sequenced and overproduced. The immediate tasks in the bacterial system are purification and characterization of metal-binding, DNA-binding and transcriptional regulation activities of the PcoR protein in vitro. The role of the copper-induced detoxification proteins is poorly understood. A systematic analysis of the chemical factors and processes involved in detoxification is proposed as the first step in elucidating the resistance mechanism. Although beyond the scope of this proposal, it is anticipated that a rigorous understanding of this mechanism of heavy metal resistance may allow the rational design of second generation antimicrobial copper and silver complexes.